Journal: Aging and Disease

Article Title: Positive Feedback Regulation of Circular RNA Hsa_circ_0000566 and HIF-1α promotes Osteosarcoma Progression and Glycolysis Metabolism

doi: 10.14336/AD.2022.0826

Figure Lengend Snippet: Hypoxia-associated circRNA profiling and expression characteristics of Hsa_circ_0000566 in osteosarcoma (OS). (A) CircRNA microarray analysis reveals 35 upregulated and 23 downregulated circRNAs in OS cells under normoxic and hypoxic conditions. The black arrow represents Hsa_circ_0000566. (B) OS cells incubated under various oxygen concentrations. Total RNA extraction was performed for qRT-PCR assay. Western blotting was performed to determine the protein level of HIF-1α. Results are reported as mean ± standard deviation (SD), *p < 0.05, n = 3. Scale bars, 200 μm. (C) Hsa_circ_0000566 expression is much higher in primary OS tissue than in chondroma tissue. Results are representative images according to three different experiments. (D) Quantitative real-time polymerase chain reaction (qRT-PCR) results comparing Hsa_circ_0000566 mRNA expression in 12 OS and chondroma samples. Results are reported as mean ± SD, *p < 0.05, n = 12. (E) Hsa_circ_0000566 expression levels in hFOB1.19 and various OS cell lines. Results are reported as mean ± SD, *p < 0.05, n = 3. (F) Schematic diagram showing Hsa_circ_0000566 back-spliced by exons 2-11 of the VRK1 gene and the corresponding Sanger sequencing. (G) RT-PCR results validating the presence of Hsa_circ_0000566 in 143B and HOS cells. Various primers amplified the Hsa_circ_0000566 region in cDNA but not in genomic DNA. β-actin was used as the negative control. Divergent primers are presented as the opposite direction of the arrowhead, and the convergent primers were shown as the face-to-face direction of the arrowhead. (H) RT-PCR results indicating Hsa_circ_0000566 and VRK1 mRNA expression in untreated 143B and HOS cells and in the cells subjected to treatment with RNase-R. (I) RNA fluorescence in situ hybridization (FISH) results revealing Hsa_circ_0000566 localized mainly in the cytoplasm. Hsa_circ_0000566 probes were labeled with cy3 and nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Scale bars, 100 μm. (J) qRT-PCR determination of the main localization of Hsa_circ_0000566 in OS cells. Results are reported as mean ± SD, *p < 0.05, n = 3.

Article Snippet: Human hFOB1.19 osteoblasts, HEK-293, and various osteosarcoma cell lines, including 143B, HOS, MG-63, and U2OS, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Expressing, Microarray, Incubation, RNA Extraction, Quantitative RT-PCR, Western Blot, Standard Deviation, Real-time Polymerase Chain Reaction, Sequencing, Reverse Transcription Polymerase Chain Reaction, Amplification, Negative Control, Fluorescence, In Situ Hybridization, Labeling, Staining

Journal: Aging and Disease

Article Title: Positive Feedback Regulation of Circular RNA Hsa_circ_0000566 and HIF-1α promotes Osteosarcoma Progression and Glycolysis Metabolism

doi: 10.14336/AD.2022.0826

Figure Lengend Snippet: Hsa_circ_0000566 contributes to in vitro osteosarcoma (OS) cell progression under hypoxic conditions. (A) Hsa_circ_0000566 overexpression and knockdown induced and repressed OS cell proliferation under hypoxia. Results are reported as mean ± standard deviation (SD), *p < 0.05, n = 3. Circ_0000566 represents Hsa_circ_0000566 overexpression, and si circ_0000566 represents Hsa_circ_0000566 knockdown. Vector and Si NC represents the negative control of Hsa_circ_0000566 overexpression and Hsa_circ_0000566 knockdown, respectively. (B) EdU exhibits the impact of Hsa_circ_0000566 on OS cell proliferation under hypoxia. Nuclei are stained with 4’,6-diamidino-2-phenylindole (DAPI). Results are reported as mean ± SD, *p < 0.05, n = 3. Scale bars, 100 μm. (C) Colony formation experiment verifies Hsa_circ_0000566 functions in OS cells under hypoxia. Results are reported as mean ± SD, *p < 0.05, n = 3. (D) Soft agar colony formation assay indicates the effects of Hsa_circ_0000566 on 143B and HOS cell colony forming capacity under hypoxia. Results are reported as mean ± SD, *p < 0.05, n = 3. Scale bars, 100 μm. (E) OS cell migration capacity as determined by Transwell™ migration assays. Results are reported as mean ± SD, *p < 0.05, n = 3. Scale bars, 100 μm. (F) Flow cytometry verifies Hsa_circ_0000566 functions in OS cell apoptosis. Results are reported as mean ± SD, *p < 0.05, n = 3.

Article Snippet: Human hFOB1.19 osteoblasts, HEK-293, and various osteosarcoma cell lines, including 143B, HOS, MG-63, and U2OS, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: In Vitro, Over Expression, Knockdown, Standard Deviation, Plasmid Preparation, Negative Control, Staining, Soft Agar Assay, Migration, Flow Cytometry

Journal: Aging and Disease

Article Title: Positive Feedback Regulation of Circular RNA Hsa_circ_0000566 and HIF-1α promotes Osteosarcoma Progression and Glycolysis Metabolism

doi: 10.14336/AD.2022.0826

Figure Lengend Snippet: Hsa_circ_0000566 accelerates osteosarcoma (OS) glucose metabolism and regulates hypoxia-enhanced glycolysis. (A) Colors of the media indicate that Hsa_circ_0000566 silencing decreased lactate accumulation under hypoxia. (B-C) Quantitative real-time polymerase chain reaction (qRT-PCR) or western blots evaluating the expression levels of genes involved in glucose metabolism in 143B and HOS cells transfected with Hsa_circ_0000566-overexpressing, Hsa_circ_0000566 (shRNA), or vector plasmids. Results are reported as mean ± standard deviation (SD), *p < 0.05, n = 3. (D) Hsa_circ_0000566 knockdown in OS cells with decreased lactate accumulation, while Hsa_circ_0000566 overexpression has increased lactate accumulation. Results are reported as mean ± SD, *p < 0.05, n = 3. (E) Extracellular acidification rate (ECAR) indicates glycolysis rate. ECAR decreases in response to Hsa_circ_0000566 knockdown and increases in response to Hsa_circ_0000566 overexpression. Oxygen consumption rate (OCR) represented mitochondrial respiratory capacity. OCR is enhanced in response to Hsa_circ_0000566 silencing and reduced in response to Hsa_circ_0000566 overexpression in OS cells. Results are reported as mean ± SD, *p < 0.05, n = 3.

Article Snippet: Human hFOB1.19 osteoblasts, HEK-293, and various osteosarcoma cell lines, including 143B, HOS, MG-63, and U2OS, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Expressing, Transfection, shRNA, Plasmid Preparation, Standard Deviation, Knockdown, Over Expression

Journal: Aging and Disease

Article Title: Positive Feedback Regulation of Circular RNA Hsa_circ_0000566 and HIF-1α promotes Osteosarcoma Progression and Glycolysis Metabolism

doi: 10.14336/AD.2022.0826

Figure Lengend Snippet: Hsa_circ_0000566 establishes interactions with HIF-1α and confers protection against ubiquitination-mediating degradation. (A) Effects of Hsa_circ_0000566 knockdown and Hsa_circ_0000566 overexpression on mRNA and protein expression in 143B and HOS cells under hypoxia. Results are reported as mean ± standard deviation (SD), *p < 0.05, n = 3. (B) Western blotting results revealing the impact of bortezomib treatment on the changes occurring at HIF-1α protein level mediated by Hsa_circ_0000566 silencing and vector transfection. (C) Western blotting assessment of the impact of CHX treatment on the variations in HIF-1α protein levels affected by Hsa_circ_0000566 silencing and vectors. Results are reported as mean ± SD, *p < 0.05, n = 3. (D) The western blot illustrates the effects of Hsa_circ_0000566 knockdown in the Hyp564 HIF-1α protein levels in the presence or absence of bortezomib treatment. (E) Immunoprecipitation assessing the HIF-1α ubiquitination levels in Hsa_circ_0000566 silencing and Hsa_circ_0000566 overexpressing osteosarcoma (OS) cells under hypoxia. Culture media were supplemented with bortezomib (250 nM) for 6 h. (F) The combination of Hsa_circ_0000566 with HIF-1α confirmed by radioimmunoprecipitation (RIP). Results are reported as mean ± SD, *p < 0.05, n = 3. (G) Pulldown assay validation of the interaction between Hsa_circ_0000566 and HIF-1α. (H) A RIP assay of HIF-1α regions interacting with Hsa_circ_0000566. Schematic diagram shows HIF-1α protein fragments. Results are reported as mean ± SD, *p < 0.05, n = 3. (I) Interaction profile between Hsa_circ_0000566 and HIF-1α obtained from catRAPID (left). (J) Schematic diagram showing Hsa_circ_0000566 RNA fragments. Combinative regions between Hsa_circ_0000566 and HIF-1α were identified by RIP assay. Results are reported as mean ± SD, *p < 0.05, n = 3.

Article Snippet: Human hFOB1.19 osteoblasts, HEK-293, and various osteosarcoma cell lines, including 143B, HOS, MG-63, and U2OS, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Ubiquitin Proteomics, Knockdown, Over Expression, Expressing, Standard Deviation, Western Blot, Plasmid Preparation, Transfection, Immunoprecipitation, Biomarker Discovery

Journal: Aging and Disease

Article Title: Positive Feedback Regulation of Circular RNA Hsa_circ_0000566 and HIF-1α promotes Osteosarcoma Progression and Glycolysis Metabolism

doi: 10.14336/AD.2022.0826

Figure Lengend Snippet: Hsa_circ_0000566 promotes osteosarcoma (OS) glucose metabolism and tumorigenesis progression in vivo. (A) 143B cells stably transfected with Hsa_circ_0000566 knockdown, HIF-1α overexpression, or empty vector plasmids. Nude mice were subcutaneously injected with 1 × 10 7 cells that were either stable negative controls or those with Hsa_circ_0000566 knockdown, HIF-1α overexpression, or Hsa_circ_0000566 knockdown. Thirty days after injection, the animals were euthanized, and their tumors dissected and photographed. (B) Tumor weight measurements on the same day the mice were euthanized. Results are reported as mean ± standard deviation (SD), *p < 0.05, n = 5. (C) Tumor volumes (ab2/2) were calculated every 6 d from the day after the mice were injected with stable OS cells. (D-E) Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) exhibit the expression levels of the genes involved in glycolysis metabolism. Results are reported as mean ± SD, *p < 0.05, n = 3. (F) Fluorescence in situ hybridization (FISH), hematoxylin and eosin (H&E) staining, and immunohistochemistry (IHC) analysis indicate the OS organization in mice and relative GLUT1, GLUT4, PDK1, PDK4, and LDHA protein levels in tumors from different groups. (G) In situ tumor formation experiment reveals that HIF-1α overexpression recovered Hsa_circ_0000566 knockdown-induced tumor attenuation. Results are reported as mean ± SD, *p < 0.05, n = 4. (H) Micro-computed tomography (CT) indicates the functions of HIF-1α and Hsa_circ_0000566 knockdown in bone loss. (I) H&E staining of lung metastasis. In mice injected in the tail vein with various stable 143B cells, lung metastasis was detected using an in vivo bioluminescence imaging system. Results are reported as mean ± SD, *p < 0.05, n = 5.

Article Snippet: Human hFOB1.19 osteoblasts, HEK-293, and various osteosarcoma cell lines, including 143B, HOS, MG-63, and U2OS, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: In Vivo, Stable Transfection, Transfection, Knockdown, Over Expression, Plasmid Preparation, Injection, Standard Deviation, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Fluorescence, In Situ Hybridization, Staining, Immunohistochemistry, In Situ, Micro-CT, Imaging

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A) Cas9/sgRNA/Oligo-targeting site near the SOX7 stop codon (underlined in red). The SgRNA sequence is highlighted in red (this contains the stop codon), while the protospacer-adjacent motif (PAM) sequence is shown in blue. The oligo donor containing the V5 tag (green box), is flanked by 60 bp homology arms. The red arrows represent PCR sequencing primers for genotyping (SF, V5R and SR). (B) PCR genotyping of CRISPR Sox7 -V5 injected live-born pups. Left: PCR genotyping with primer pair SF and V5R showed the desired band at the correct size in postnatal (P) 3, P4, P6, P7 and P10. Right: PCR genotyping with primer pair SF and SR produced slightly larger products in P4, P6 and P10 compared to P3 and P7, indicating successful 42 bp V5 tag integration. At least 3 bands were discovered in P3 and P7, but not in P4, P6 and P10 when separated by 3.5% gel electrophoresis (not shown). Red arrows indicate the expected PCR product sizes. (C) Sequencing of PCR product using SF primer (for both SF/V5R and SF/SR) confirmed the correct fusion of V5 tagged to the last codon in both Sox7 alleles of the Sox7 -V5 founders.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Sequencing, CRISPR, Injection, Produced, Nucleic Acid Electrophoresis

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A) At E10.5, SOX7 (white) is expressed in intersomitic vessels (ISVs), the dorsal aorta (DA) (delineated by arterial marker, SOX17), cardinal vein (CV), and the surrounding migrating endothelial cells (red arrowheads) (B) SOX7 was not detected in PROX1+ migrating lymphatic endothelial cells (LECs) (asterisks). (C) At E16.5, SOX7 is downregulated in the SOX17+ main arterial branch (boxed area with dotted line), but continues to be expressed in the less mature arteries near the midline (box area with solid line). Arteries are delineated by arterial marker SOX17; major veins and vascular plexus are delineated by endomucin (EMCN). (D) SOX7/ β -galactosidase ( β -gal) (green) also stains the SOX17 (white) positive major arteries in the skin plexus of E14.5 Sox7 +/- lac-Z reporter mice. Dors., dorsal; Med., medial. Scale bars = 100 μm.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Marker

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A) Representative images of E10.5 Sox7 iECKO mutants and sibling controls, injected with tamoxifen at E9.5. The endothelium is delineated by endomucin (EMCN) (blue) and endothelial cells by ETS-related gene (ERG) (red). (Top right panel) The percentage of SOX7 positive endothelial cells over the total number of endothelial cells quantified from 10 sequential transverse sections in 2 control and 3 Sox7 iECKO mutants. (Bottom right panel) Graph indicating the levels of Sox7 transcript in sibling controls and Sox7 iECKO mutants. Mean ± SEM; scored sibling control, n=9; Sox7 iECKO mutants, n=7; Mann-Whitney U- test. P <0.0005 (***). (B) Bright-field images of Sox7 iECKO mutants and sibling controls at E14.5, after pulsing with tamoxifen at E9.5 and E10.5. Scored sibling controls, n=8; Sox7 iECKO mutants, n=5. (C) Brightfield images and whole-mount immunostaining of Sox7 iECKO mutant and sibling control skin at E13.5, after Cre induction at E9.5 and E10.5. Dermal lymphatic structures are marked by Neuropilin 2 (NRP2) (membranous white), lymphatic endothelial cells by Prospero-related homeobox 1 (PROX1) (red), and blood vessels by EMCN (green). Dash line represents the midline of the embryo. Scored sibling controls, n=7; Sox7 iECKO mutants, n=7 (D-H) Quantification of (D) lymphatic sprout migration distance from the midline (E) lymphatic branch points/area (F) lymphatic vessel width (μm) (G) PROX1+ nuclei/area and (H) PROX1+ nuclei/lymphatic vessels across the whole skin. Total area = 4200 x 1500 μm on both sides of the midline, in sibling controls (n=3) and Sox7 iECKO mutants (n=5-6). (F,H) Average of width and PROX1+ nuclei was obtained from 7 random representative leading lymphatic vessels, of fixed length at 200 μm, from both sides of the midline in each skin. (I,J) Quantification of (I) disconnected lymphatic endothelial cell (LEC) clusters (<100 μm) and vessel branches (>100μm)/area and (J) PROX1+ nuclei in each LEC cluster. Total area quantified = 4200 x 2200 μm on both side of midline in sibling controls (n=3) and Sox7 iECKO mutants (n=5). PROX1+ nuclei were quantified from n=29 and n=122 LEC clusters, respectively. (D-J) Skins were from the cervico-thoracic regions of E13.5 embryos, defloxed at E9.5, E10.5. Mean ± SEM, Mann-Whitney U –test. P <0.05 (*). LV, lymphatic vessel. Scale bars = 100 μm (immunofluorescence images A,C), 1 mm (bright-field images B,C).

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Injection, MANN-WHITNEY, Immunostaining, Mutagenesis, Migration, Immunofluorescence

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A) Whole-mount immunostaining of Sox7 iECKO mutant and sibling control embryonic skin at E14.5, following injection with tamoxifen at E9.5 and E10.5. Dermal lymphatic structures are marked by NRP2 (membranous white), lymphatic endothelial cells by PROX1 (red), blood vessels by EMCN (green) and arterioles by SOX7 (white nuclei). Dashed line represents the midline of the embryo. (B) Whole-mount immunostaining of Sox7 iECKO mutant and sibling control for EMCN (grey), showing dermal blood vessels at E13.5, after injection with tamoxifen at E9.5 and E10.5. Endothelial cells are marked by ERG (red), proliferative cells are stained by phospho-histone 3, H3 (green). (Right panel) Quantitation of EMCN+ (blood) vessel front density (μm 3 ), number of H3+ proliferative and ERG+ endothelial cells in this region. Scored sibling control, n=5; Sox7 iECKO mutants, n=4; Mean ± SEM; Mann-Whitney U -test. P <0.05 (*); ns = not significant. (C) Whole-mount immunostaining of Sox7 iECKO ; mT/mG mutant and Cre+; mT/mG control embryonic skin at E14.5, after injection with tamoxifen at E12.5. Blood vessels are stained with EMCN (red), cells after Cre excision are shown in green, and lymphatic endothelial cells are marked by PROX1 (white). (D-F) Graphs showing quantitation of the Cre activity in (D) blood vessels, (E) lymphatic vessels and (F) EMCN+ (blood) vessel density (μm 3 ), from n=4 Cre+; mT/mG controls and n=3 Sox7 iECKO ; mT/mG mutants. Mean ± SEM; Mann-Whitney U -test; ns = not significant. (G) Whole-mount immunostaining of Sox7 iECKO mutant and sibling control embryonic skin at E14.5, after injection with tamoxifen at E11.5 and E12.5. Dermal lymphatic structures are marked by the NRP2 (membranous white), lymphatic endothelial cells by PROX1 (red), and blood vessels are stained by EMCN (green). Dashed line represents the midline of the embryo. (H) Immunostaining of Sox7 iECKO mutant and sibling control coronal sections, at E11.5, after injection with tamoxifen at E9.5, E10.5. Lymphatic progenitor cells in the cardinal veins (CVs) are PROX1+ (white). Yellow arrowheads highlight the presence of LEC progenitors in the region near the dorsal aorta (DA) in the Sox7 iECKO mutant, which is typically absent in the sibling control. Scale bars = 100 μm.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Immunostaining, Mutagenesis, Injection, Staining, Quantitation Assay, MANN-WHITNEY, Activity Assay

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A-B) Nebulosa plots from single-nuclei RNA-Seq on E14.5 embryonic skins, showing the expression of Sox7 and Vegfc. Both Sox7 and Vegfc are found expressed in BECs (red arrowheads), but not LECs (empty red arrowheads). (C) Vegfc (red) fluorescent RNA in situ hybridisation on mouse cross-sections at E11.5. The endothelium is delineated by PECAM (white) and LECs by PROX1 (green nuclei). BEC-specific endogenous levels of Vegfc (red arrowheads) are higher than LECs (asterisks). (D) qPCR on FACs-sorted PECAM+CD45- endothelial cells of Sox7 iECKO mutants and sibling controls at E14.5, injected with tamoxifen at E11.5 and E12.5. Expression is normalised to endothelial marker Pecam and Cdh5 . Scored sibling controls, n=9; Sox7 iECKO mutants, n=8. (E-F) qPCR on human arterial endothelial cells (HUAECs) transfected with SiSOX7 or SiCTRL for (E) 30 h and (F) 17 h. (G) qPCR on human venous endothelial cells (HUVECS) transfected with SiSOX7 or SiCTRL for 17 h. (E-G) Expression is relative to HPRT and GAPDH. Data from one siRNA experiment performed in triplicates. (D–G) Mean ± SEM; t -test. P <0.05 (*); P <0.005 (**); P <0.0005 (***). BEC, blood endothelial cell; LEC, lymphatic endothelial cell; DA, dorsal aorta; CV, cardinal vein; Dors., dorsal; Med., medial. Scale bars = 100 μm.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: RNA Sequencing Assay, Expressing, In Situ, Hybridization, Injection, Marker, Transfection

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A-D) Nebulosa plots from single-nuclei RNA-Seq on E14.5 embryonic skins, showing markers for BECs ( Emcn ), LECs ( Prox1 ), SMCs ( Acta2 ) and neuronal cells ( Npas3 ). (E) Microarray analysis of dermal BEC and LEC populations sorted from different embryonic stages reveals that Sox7 and Vegfc mRNA are preferentially expressed by BECs. BECs, blood endothelial cells; LECs, lymphatic endothelial cells; and SMC, smooth muscle cells.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: RNA Sequencing Assay, Microarray

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A-D) SOX7 transcriptionally activates Notch effector, HEY1, to repress Vegfc . (A) qPCR on FAC-sorted PECAM+CD45- endothelial cells of Sox7 iECKO mutants and sibling controls at E14.5, injected with tamoxifen at E11.5 and E12.5. Expression is normalised to the endothelial marker Pecam and Cdh5 . Scored sibling controls, n=9; Sox7 iECKO mutants, n=8. (B-C) qPCR on (B) human arterial endothelial cells (HUAECs) and (C) human venous endothelial cells (HUVECs) transfected with SiSOX7 or SiCTRL for 17 h. In addition to HEY1, DLL4 levels were also downregulated in the human cell line experiments. Expression is relative to HPRT and GAPDH. Data from one siRNA experiment performed in triplicates. Mean ± SEM; t -test. P <0.05 (*); P <0.005 (**); P <0.0005 (***). (D) HEY1 represses human VEGFC promoter activity. HeLa cells were co-transfected with human or mouse VEGFC-luc and either EV (empty vector) or HEY1 expression constructs as indicated. VEGFC luciferase activity was measured and normalised to Renillla luciferase activity, which was then made relative to the promoter-less vector, pGL3-basic, which was set to 1. Biological replicates, n=3 independent repeats of the same experiment. Mean ± SEM; t -test. P <0.05 (*). (E-G) SOX7 physically interacts with transcription repressor, HEY1. (E) Amplified Luminescent Proximity Homogenous Assay (ALPHAScreen) shows the heatmap of SOX7 pairwise protein-protein interaction tested, where red indicates strong interaction and light blue indicates an absence of interaction. (F) Single molecule spectroscopy reveals that SOX7 is able to directly interact with HEY1. SOX7 and the target transcription factors were tagged with GFP or Cherry. GFP-SOX7 and HEY1-Cherry show co-incidence at 0.66, suggesting a 1:2 interaction. Peaks centered on 0 (green) or 1 (red) correspond to the GFP or Cherry-tagged proteins only. (G) Co-immnunoprecipitation analysis of SOX7 and HEY1. HEK293 cells were transfected with indicated plasmids and harvested 24 h after transfection, immunoprecipitated by anti-GFP or Ig control before immunoblot analysis to determine the presence of bait/prey (top) and the input (bottom). N=2.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Injection, Expressing, Marker, Transfection, Activity Assay, Plasmid Preparation, Construct, Luciferase, Amplification, Amplified Luminescent Proximity Homogenous Assay, Spectroscopy, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A) SOX7 represses human VEGFC promoter activity. HeLa cells were co-transfected with human or mouse VEGFC-luc, EV (empty vector), or SOX7 expression constructs as indicated. VEGFC luciferase activity was measured and normalised to Renillla luciferase activity, which was then made relative to the promoter-less vector, pGL3-basic, which was set to 1. Biological replicates, n=3. Mean ± SEM; t -test. P <0.05 (*). (B) Schematic representation of the human VEGFC locus 255 kb upstream from the transcription start site (TSS) (denoted as VEGFC-255 region) from the UCSC Genome browser. The H3K27Ac is denoted in light blue, DNAseI hypersensitive hotspots are indicated by black/grey boxes, where the darkness is proportional to the maximum signal strength observed in any cell line. The chromatin state in HUVECs is shown in purple (indicates insulator), green (poised enhancer) and pink (a Polycomb-repressed region). The HUVECs CCCTC-binding factor (CTCF) binding location is shown in orange. Multiple species conservation is shown in blue peaks and alignments with black stripes. (C) The human luciferase VEGFC-255 BCR transgene. B (black), binding region; C (blue), conserved region and R (pink), repressive region. (D) Schematic representation of the mouse Vegfc locus 152kb upstream from the TSS (denoted as Vegfc- 152). The chromatin state in mouse at E11 from ENCODE is denoted in pink (indicates a heterochromatin region enriched for H3K9me3 repressive marker). The ATAC-Seq (in black) indicates accessible DNA regions in E11 mouse hearts. Multiple species conservation is shown in blue peaks and alignments with black stripes. (E) The mouse luciferase Vegfc-152 RBC transgene. R (pink), repressive region; B (black), binding region and C (blue), conserved region. (F) SOX7 represses VEGFC promoter activity through VEGFC-255 and Vegfc-152 . HeLa cells were co-transfected with human or mouse VEGFC-luc, VEGFC-255 BC-luc, VEGFC-255 BCR-luc, Vegfc-152 BC-luc, Vegfc-152 RBC-luc, EV (empty vector), or SOX7 expression constructs as indicated. VEGFC luciferase activity was measured and normalised to Renillla luciferase activity, which was then made relative to the promoter-less vector, pGL3-basic, which was set to 1. Biological replicates, n=3. Mean ± SEM; t -test. P <0.05 (*); P <0.005 (**); ns = not significant.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, Construct, Luciferase, Binding Assay, Marker

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A) MSigDB pathway analysis on the top 2k peaks from human SOX7 HUVEC ChIP-Seq identified enrichment for genes within the VEGF/VEGFR gene set. These include VEGFR1 , VEGFR2 , NRP1 , NRP2 , PDGFC , VEGFA and VEGFC . (B-F) Binding profiles and motif analysis of mouse SOX7-V5 ChIP-Seq (B) Image from Integrative Genome Viewer (IGV) illustrating ChIP-Seq tracks at E9.5 (pink) and E10.5 (light blue) mapped to the mouse reference genome GRCm38/mm10 (top track, dark blue). The height of a graph peak indicates the number of “positive bindings” on a given chromosomal location. Total number of binding events is indicated on the left under each track name. Top DNA-binding motifs in SOX7 - V5 ChIP-Seq by MEME-ChIP in (C) E9.5 and (D) E10.5 embryos. The different height of the letters in the position weight matrix reveals information content of each position (in bits), correlated by the degree of certainty of the nucleotide at a given position. (E) Spaced motif analysis (SPAMO) found a SOX7 primary motif amongst the topmost enriched motif next to a MEF2A/MEF2C binding motif found in E9.5 SOX7 - V5 ChIP-Seq, with 2093 total occurrences. SOX7 is best gapped at 134 bp from the MEF2A/MEF2C binding motif (24/2093). (F) ChIP-PCR on both bound fraction (left) and corresponding assumed unbound controls (right), as predicted by the peak location from SOX7-V5 ChIP-Seq. (G) A total of 712 genes were found overlapping between E9.5 and E10.5 SOX7-V5 mouse ChIP-Seq. (H-J) KEGG pathway analysis (H) and gene ontological analysis by DAVID bioinformatics database, showing the top biological processes (I) and molecular functions (J) associated to the 712 overlapping genes from the SOX7-V5 mouse ChIP-Seq.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A) At 2 days post Cre induction, E11.5 Sox7 iECKO mutants showed ectopic expression of PROX1+ (red) in medial ventral regions of the cardinal veins (CVs), close to dorsal aorta (DA) (yellow arrowheads). (B) Rose diagram indicates the % of PROX1+ cells over total number of EMCN+ endothelial cells, within each indicated region. The medial region closest to the DA is indicated by 0/360°; most lateral region, 180°; dorsal, 90° and ventral, 270°. Scored sibling controls, n=5; Sox7 iECKO mutants, n=4. Mean ± SEM; Mann-Whitney U -test. P <0.05 (*). (C) Graph indicates the % of total PROX1+ cells over the total number of EMCN+ endothelial cells within each embryo analysed. A total of 2907 endothelial cells quantified, from n=5 sibling controls and 1780 endothelial cells from n=4 Sox7 iECKO mutants. Mean ± SEM; Mann-Whitney U -test. P =0.0635. (D) Whole-mount immunostaining of control and Sox7 iECKO embryonic skin at E12.5 after Cre induction at E9.5, E10.5. Dermal lymphatic structures are stained with NRP2 (membranous white), lymphatic endothelial cells, PROX1 (red), and blood vessels, both EMCN (green) and SOX7 (white nuclei). Arrowheads indicate EMCN low/- PROX1 + NRP2 +/low individual LECs on blood vessels (type I), and arrow shows EMCN + PROX1 + NRP2 low/- LEC within the vessel wall (type II). (E) Graph shows the total events of PROX1+ single LECs or LEC clusters cells associated with EMCN+ blood vessel plexus within each embryo. PROX1+ cells were quantified from n=4 controls and n=5 Sox7 iECKO mutants. Mean ± SEM; Mann-Whitney U -test. P<0.05 (*). Dors., dorsal; Med., medial. Scale bars = 100 μm.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Expressing, MANN-WHITNEY, Immunostaining, Staining

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A-B) Whole-mount immunostaining of Sox7 iECKO mutant or control embryonic skin, showing the different types of LECs emerging from the blood capillary plexus. Skins were harvested from embryos at E12.5, after Cre-induction at E9.5 and E10.5. Dermal lymphatic structures are marked by NRP2 (membranous white), lymphatic endothelial cells by PROX1 (red), and blood vessels are stained by EMCN (green) and SOX7 (white nuclei). Arrowheads indicate EMCN low/- PROX1 + NRP2 +/low individual LECs on blood vessels (type I), arrows show the single EMCN + PROX1 + NRP2 low/- LEC within the blood vessel wall (type II) and empty arrowheads mark the EMCN +/- PROX1 + NRP2 +/low LEC clusters (2-4 cells) exiting from the blood vessel network (type III). (C-E) Graph shows the quantitation of different types of PROX1+ LEC progenitors, comparing between control and Sox7 iECKO mutant skins. PROX1+ LEC progenitors were quantified from n=4 controls and n=5 Sox7 iECKO mutants. Mean ± SEM; Mann-Whitney U -test. P <0.05 (*); ns = not significant. Scale bars = 100 μm.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Immunostaining, Mutagenesis, Staining, Quantitation Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A) Table from HOMER motif database showing the top 10 most enriched transcription motifs associated to SOX7 binding sites (B, G) Graphs showing intersections between top 2k SOX7 ChIP-Seq peak locations and HUVEC or mouse histone markers, respectively from the ENCODE consortium. Only peaks with at least 50% overlapping region with the histone markers are considered. Intersection was performed using the EpiExplorer online resource tool. (C, H, J) Venn diagrams showing intersections of different active histone markers and (D, I, K) repressive histone markers, both displayed at least 50% overlapped, with the top 2k of SOX7 ChIP-Seq binding regions. Diagram was generated using online resource stool, Venny 2.1.0. (E, L) Graphs showing the overall proportion of top 2k SOX7 ChIP-Seq peaks, with at least an active or a repressive histone mark. (F) Graphs showing intersections between top 2k SOX7 HUVEC ChIP-Seq peak locations and chromatin states in HUVECs from the ENCODE consortium. Only peaks with at least 50% overlapping region with each corresponding chromatin state are considered. Intersection was performed using the EpiExplorer online resource tool.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Binding Assay, ChIP-sequencing, Generated

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A and B) LEC progenitor organisation is impaired in both early stage and organ-specific lymphangiogenesis. ( A ) In physiological conditions, LEC progenitors emerge in the dorsal lateral part of the CVs and migrate towards the dorso-lateral aspects of the embryo. In the absence of functional SOX7, blood vascular endothelial cells from the arterial compartment increase the local expression of endothelial VEGFC, perturbing its tissue distribution. This induces and/or expands the number of LEC progenitors in ventro-medial aspects of the CVs. ( B ) Dermal lymphatics are dysmorphic in the absence of SOX7 function in blood vascular endothelial cells. An increase in VEGFC in the dermal blood endothelial cells causes hyperproliferation of local LEC progenitors. This is shown by an increase in the emergence of local LEC progenitors from the endomucin-positive blood capillary plexus at E12.5 (red asterisks). Changes in expressions of other SOX7-dependent lymphangiocrines also contribute to the migration defects in the dermal lymphatics. ( C ) Model showing the molecular mechanisms of SOX7-dependent repression of VEGFC transcription identified in this study: 1) SOX7 binds to distal regulatory elements (silencing regions) to suppress VEGFC transcription. 2) SOX7 represses VEGFC promoter activity. 3) SOX7 acts upstream of Notch effector and repressor, HEY1. SOX7-induced expression of HEY1 causes VEGFC downregulation and 4) Protein-protein interaction between SOX7 and HEY1 forms a complex to repress VEGFC transcription. LEC, lymphatic endothelial cells; AEC, arterial endothelial cells; VEC, venous endothelial cells; Lat., lateral; Med, medial.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Functional Assay, Expressing, Migration, Activity Assay

Journal: BMC Evolutionary Biology

Article Title: Suboptimal evolutionary novel environments promote singular altered gravity responses of transcriptome during Drosophila metamorphosis

doi: 10.1186/1471-2148-13-133

Figure Lengend Snippet: Description of the 90 microarray CEL files used (accession number GEO NCBI database GSE33779 )

Article Snippet: ILV performed RNA amplification and Affymetrix microarray hybridizations and preliminary data analysis.

Techniques: Microarray

Journal: bioRxiv

Article Title: Identification, Isolation, and Characterization of Human LGR5-positive Colon Adenoma Cells

doi: 10.1101/118034

Figure Lengend Snippet: (A) LGR5 IHC staining in normal human colon (one of five representative patients) at low (A1) and high (A2) magnification, as well as adenoma (A3) from the same patient (high-grade dysplasia; adjacent to adenocarcinoma; specimen 14881). (B) Lgr5 expression by in situ hybridization provides a conventional reference for the LGR5 IHC staining in normal crypts (upper panel) and in the adenoma (bottom panel); glandular Lgr5 (arrow-1) and stromal expression (arrow-2) in adenoma; (C) LGR5 IHC (C1, C2) and IF staining (C3, C4) in fetal duodenum, and (D) ISH expression in the same duodenum specimen. Scale bars, 100μM; A2, 25μM.

Article Snippet: Flow cytometry spike-in experiments ( ) were performed with 1881 LGR5(+) and 1881 LGR5(−) by mixing at varying proportions and analyzing on a LSRII cytometer (BD Biosciences) before and after magnetic separation with rat monoclonal antibody anti-human LGR5 clone 22H2.8 magnetic bead-conjugate (Miltenyi Biotec), with the allophycocyanin (APC) anti-bead check reagent to recognize the bound magnetic beads (Miltenyi Biotec).

Techniques: Immunohistochemistry, Expressing, In Situ Hybridization, Staining

Journal: bioRxiv

Article Title: Identification, Isolation, and Characterization of Human LGR5-positive Colon Adenoma Cells

doi: 10.1101/118034

Figure Lengend Snippet: (A) LGR5 immunohistochemical and immunofluorescent staining in adult duodenum showed weak punctate LGR5 expression, while adjacent cells stained with the paneth cell marker DefensinA5 (DEFA5) (middle and right panel). Scale bars, 50μM. (B) Mouse 1881 lymphoma cells were previously transfected with human Lgr5 [1881(+); Miltenyi Biotec]. To confirm transfection stability, the 1881 LGR5(+) and 1881 Lgr5(−) cells were assayed for Lgr5 expression (n=3 biological replicates), and visualized with agarose gel analysis of qRT-PCR amplified products. Line represents mean values. (C) Measurement of human LGR5 protein expression by Western blotting with the rabbit monoclonal anti-human LGR5 antibody clone STE- 1-89-11.5 in 1881(+) cells, 282 adenoma organoids cultured in KGMG, and 14881 adenoma organoids cultured in KGMG or L-WRN.

Article Snippet: Flow cytometry spike-in experiments ( ) were performed with 1881 LGR5(+) and 1881 LGR5(−) by mixing at varying proportions and analyzing on a LSRII cytometer (BD Biosciences) before and after magnetic separation with rat monoclonal antibody anti-human LGR5 clone 22H2.8 magnetic bead-conjugate (Miltenyi Biotec), with the allophycocyanin (APC) anti-bead check reagent to recognize the bound magnetic beads (Miltenyi Biotec).

Techniques: Immunohistochemical staining, Staining, Expressing, Marker, Transfection, Agarose Gel Electrophoresis, Quantitative RT-PCR, Amplification, Western Blot, Cell Culture

Journal: bioRxiv

Article Title: Identification, Isolation, and Characterization of Human LGR5-positive Colon Adenoma Cells

doi: 10.1101/118034

Figure Lengend Snippet: (A) Representative images of LGR5 staining in the epithelial and stromal components in normal colon (n=5 normal autopsy samples) and from a CRC tissue microarray (n=2 normals, 65-68 neoplasm). Scale bar, 50μM. (B) Association between epithelial or (C) stromal staining intensity, and stage/grade of the tissue tested by generalized linear modeling, with staining intensity as the dependent variable and stage or grade as the independent variable.

Article Snippet: Flow cytometry spike-in experiments ( ) were performed with 1881 LGR5(+) and 1881 LGR5(−) by mixing at varying proportions and analyzing on a LSRII cytometer (BD Biosciences) before and after magnetic separation with rat monoclonal antibody anti-human LGR5 clone 22H2.8 magnetic bead-conjugate (Miltenyi Biotec), with the allophycocyanin (APC) anti-bead check reagent to recognize the bound magnetic beads (Miltenyi Biotec).

Techniques: Staining, Microarray

Journal: bioRxiv

Article Title: Identification, Isolation, and Characterization of Human LGR5-positive Colon Adenoma Cells

doi: 10.1101/118034

Figure Lengend Snippet: (A) Graphical representation of complete procedure. (B) Culture of adenoma organoids for LGR5 enrichment. LGR5 IHC staining (left column) in biopsied large adenoma (>10mm; two representative specimens are shown). Organoids with budding morphology (middle column) derived from these specimens cultured in the reduced medium KGMG. The same cultures with cystic morphology (right column) after being transferred for 3-4 weeks to L-WRN medium. Scale bars, 100μM. (C) Representative scatterplots of LGR5 expression in organoids cultured in KGMG or L-WRN (specimen 282). Live, LGR5(+) human adenoma cells were obtained after LGR5-magnetic bead enrichment by isolating DAPI(−) and LGR5(+) cellular populations.

Article Snippet: Flow cytometry spike-in experiments ( ) were performed with 1881 LGR5(+) and 1881 LGR5(−) by mixing at varying proportions and analyzing on a LSRII cytometer (BD Biosciences) before and after magnetic separation with rat monoclonal antibody anti-human LGR5 clone 22H2.8 magnetic bead-conjugate (Miltenyi Biotec), with the allophycocyanin (APC) anti-bead check reagent to recognize the bound magnetic beads (Miltenyi Biotec).

Techniques: Immunohistochemistry, Derivative Assay, Cell Culture, Expressing

Journal: bioRxiv

Article Title: Identification, Isolation, and Characterization of Human LGR5-positive Colon Adenoma Cells

doi: 10.1101/118034

Figure Lengend Snippet: (A) Relative LGR5 , SMOC2 , and DKK4 mRNA expression in L-WRN vs KGMG adenoma organoid cultures. Individual specimen qRT-PCR technical replicates; represents the mean±s.e.m. of n=3 biological replicates. (B) LGR5 immunohistochemistry staining and (C) SMOC2 and Lgr5 mRNA in situ hybridization (ISH) of formalin fixed paraffin-embedded (FFPE) adenoma 282 organoids cultured in L- WRN. Arrows designate localization of Lgr5 and SMOC2 in corresponding regions of consecutive serial sections. Scale bar, 50μM. (D) DKK4 and Lgr5 ISH images in consecutive FFPE cuts of a colon adenocarcinoma (specimen 815). Scale bar, 50μM.

Article Snippet: Flow cytometry spike-in experiments ( ) were performed with 1881 LGR5(+) and 1881 LGR5(−) by mixing at varying proportions and analyzing on a LSRII cytometer (BD Biosciences) before and after magnetic separation with rat monoclonal antibody anti-human LGR5 clone 22H2.8 magnetic bead-conjugate (Miltenyi Biotec), with the allophycocyanin (APC) anti-bead check reagent to recognize the bound magnetic beads (Miltenyi Biotec).

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemistry, Staining, In Situ Hybridization, Formalin-fixed Paraffin-Embedded, Cell Culture

Journal: bioRxiv

Article Title: Identification, Isolation, and Characterization of Human LGR5-positive Colon Adenoma Cells

doi: 10.1101/118034

Figure Lengend Snippet: (A) Flow cytometric analysis for enhanced number and fluorescent intensity of LGR5(+) cells resulting from organoids cultured in the reduced medium KGMG and then transferred for 34 weeks to L-WRN medium. (B) Flow cytometry spike-in experiments to confirm LGR5 antibody specificity and to validate the magnetic bead enrichment strategy. (1) Pure populations of 1881 LGR5(+) and 1881 Lgr5(−) cells were analyzed by flow to establish gating strategy. 1881 LGR5(+) and 1881 Lgr5(−) cells were mixed in the proportions, (2) 50/50 and (3) 5/95; and analyzed by flow cytometry before (left column) and after magnetic separation (middle/right columns) with rat monoclonal anti-human LGR5 antibody clone 22H2.8.

Article Snippet: Flow cytometry spike-in experiments ( ) were performed with 1881 LGR5(+) and 1881 LGR5(−) by mixing at varying proportions and analyzing on a LSRII cytometer (BD Biosciences) before and after magnetic separation with rat monoclonal antibody anti-human LGR5 clone 22H2.8 magnetic bead-conjugate (Miltenyi Biotec), with the allophycocyanin (APC) anti-bead check reagent to recognize the bound magnetic beads (Miltenyi Biotec).

Techniques: Cell Culture, Flow Cytometry

Journal: bioRxiv

Article Title: Identification, Isolation, and Characterization of Human LGR5-positive Colon Adenoma Cells

doi: 10.1101/118034

Figure Lengend Snippet: (A) Multidimensional scaling plot of the LGR5(+) and LGR5(−) cells, based on the top 500 most variable genes. Patient identifiers #14881, 228, 584, and 590. (B) False discovery rate (FDR) volcano plot of the log(2) ratio of gene expression between the LGR5(+) and LGR5(−) cells. (C) Unsupervised hierarchical clustering heatmap of the 50 most differentially expressed genes by fold change between the LGR5(+) (red) and LGR5(−) (green) populations. (D) Log(2) fold change in gene expression between LGR5(+) and LGR5(−) cells for known markers of colon stem (red) and differentiated (green) cells. (E) The top 10 most enriched KEGG pathways for differentially expressed genes between the LGR5(+) and Lgr5(−) cells.

Article Snippet: Flow cytometry spike-in experiments ( ) were performed with 1881 LGR5(+) and 1881 LGR5(−) by mixing at varying proportions and analyzing on a LSRII cytometer (BD Biosciences) before and after magnetic separation with rat monoclonal antibody anti-human LGR5 clone 22H2.8 magnetic bead-conjugate (Miltenyi Biotec), with the allophycocyanin (APC) anti-bead check reagent to recognize the bound magnetic beads (Miltenyi Biotec).

Techniques: Gene Expression

Journal: bioRxiv

Article Title: Identification, Isolation, and Characterization of Human LGR5-positive Colon Adenoma Cells

doi: 10.1101/118034

Figure Lengend Snippet: (A) Multidimensional scaling plot of the LGR5(+), sorted MACS magnet-bound Lgr5(−) cells, and sorted MACS flowthrough LGR5(FT-) cells based on the top 500 most variable genes. (B) Unsupervised hierarchical clustering heatmap of the 50 most variable genes between the LGR5(+) (red), Lgr5(−) (green), and LGR5(FT-) (blue) populations. (C) The top 10 most enriched KEGG pathways for differentially expressed genes between the LGR5(+) and Lgr5(−); and LGR5(+) and LGR5(FT-) cells. (D) Gene expression overlap between the [LGR5(+) vs. Lgr5(−)], [LGR5(+) vs. LGR5(FT-)], and [Lgr5(−) vs. LGR5(FT-)].

Article Snippet: Flow cytometry spike-in experiments ( ) were performed with 1881 LGR5(+) and 1881 LGR5(−) by mixing at varying proportions and analyzing on a LSRII cytometer (BD Biosciences) before and after magnetic separation with rat monoclonal antibody anti-human LGR5 clone 22H2.8 magnetic bead-conjugate (Miltenyi Biotec), with the allophycocyanin (APC) anti-bead check reagent to recognize the bound magnetic beads (Miltenyi Biotec).

Techniques: Gene Expression

Journal: bioRxiv

Article Title: Identification, Isolation, and Characterization of Human LGR5-positive Colon Adenoma Cells

doi: 10.1101/118034

Figure Lengend Snippet: (A) Concordance of adenoma LGR5(+) cell gene expression with gene expression associated with Wnt signaling in colon cancer stem cells, based on TOP-GFP Wnt reporter activity . (B) Brightfield and fluorescence images of the 282 adenoma organoid line lentivirally transduced with a TCF/LEF -GFP reporter for Wnt signaling. Scale bar, 50μM. (C) Representative ImageStream flow cytometry results of TCF/LEF -GFP organoids stained with the LGR5- APC antibody (n=3 experimental replicates). Scale bar, 10μM. (D) Comparison of LGR5 expression in the 10% highest and 10% lowest TCF/LEF -GFP expressing cells; values in iii and iv represent mean % ± s.e.m.

Article Snippet: Flow cytometry spike-in experiments ( ) were performed with 1881 LGR5(+) and 1881 LGR5(−) by mixing at varying proportions and analyzing on a LSRII cytometer (BD Biosciences) before and after magnetic separation with rat monoclonal antibody anti-human LGR5 clone 22H2.8 magnetic bead-conjugate (Miltenyi Biotec), with the allophycocyanin (APC) anti-bead check reagent to recognize the bound magnetic beads (Miltenyi Biotec).

Techniques: Gene Expression, Activity Assay, Fluorescence, Transduction, Flow Cytometry, Staining, Comparison, Expressing

Journal: bioRxiv

Article Title: Identification, Isolation, and Characterization of Human LGR5-positive Colon Adenoma Cells

doi: 10.1101/118034

Figure Lengend Snippet: (A) Gene expression overlap between genes upregulated in LGR5(+) vs. LGR5(−) cells and the Lgr5 mouse intestinal stem cell signature reported in Munoz et al . (B) Comparison of gene expression between LGR5(+) adenoma cells and previously published RNA-seq data from normal human colon for genes in the “human adenoma LGR5+ cell gene signature. (C) The top ten most differentially expressed genes by magnitude between LGR5(+) adenoma cells and normal human colon (all genes; unbiased analysis) . (D) An analysis of TCGA colorectal cancer gene expression data through Oncomine™ of DKK4, comparing colorectal adenocarcinoma expression with normal colon and rectum.

Article Snippet: Flow cytometry spike-in experiments ( ) were performed with 1881 LGR5(+) and 1881 LGR5(−) by mixing at varying proportions and analyzing on a LSRII cytometer (BD Biosciences) before and after magnetic separation with rat monoclonal antibody anti-human LGR5 clone 22H2.8 magnetic bead-conjugate (Miltenyi Biotec), with the allophycocyanin (APC) anti-bead check reagent to recognize the bound magnetic beads (Miltenyi Biotec).

Techniques: Gene Expression, Comparison, RNA Sequencing, Expressing

Journal: bioRxiv

Article Title: Identification, Isolation, and Characterization of Human LGR5-positive Colon Adenoma Cells

doi: 10.1101/118034

Figure Lengend Snippet: (A) Flow cytometry gating strategy for LGR5(+) cells isolated directly from warm autopsy normal colon tissue (specimen 84, a representative scatter plots of 3 patients; ). Includes EpCAM(+) inclusion criteria, using a phycoerythrin (PE)-conjugated antibody (not shown). To set the LGR5-APC(+) gating, either magnet flow-through (specimen 81 & 83), as done with organoid sorts, or rigorous so-called fluorescence- minus-one controls [FMO; DAPI(−), EpCAM(+); specimen 84] were used. (B) mRNA expression by qRT-PCR of Lgr5 and OLFM4 across all 3 patient samples. Values represent mean ± SE. (C) OLFM4 immunofluorescent staining of normal colon crypts. Scale bar, 100μM. (D) Normal colon organoid cultures were established from warm autopsy resections. LGR5(+)/(−) cells were isolated from early passage organoids, sorted into culture medium and seeded into Matrigel to access organoid-forming efficiency. Mean organoid-forming efficiency from two normal colon organoid cultures (specimen 78, passage 5; and specimen 85, passage 3) sorted for Lgr5(−) vs. LGR5(+) cells (n=4 wells per fraction), error bars represent s.e.m. Representative images (right panels; specimen 85) of organoids formed from Lgr5(−) and LGR5(+) cells shown at day 14 and day 52 (passage 5). Scale bars, 200μM top panel; 1mm bottom panel.

Article Snippet: Flow cytometry spike-in experiments ( ) were performed with 1881 LGR5(+) and 1881 LGR5(−) by mixing at varying proportions and analyzing on a LSRII cytometer (BD Biosciences) before and after magnetic separation with rat monoclonal antibody anti-human LGR5 clone 22H2.8 magnetic bead-conjugate (Miltenyi Biotec), with the allophycocyanin (APC) anti-bead check reagent to recognize the bound magnetic beads (Miltenyi Biotec).

Techniques: Flow Cytometry, Isolation, Fluorescence, Expressing, Quantitative RT-PCR, Staining

Journal: bioRxiv

Article Title: Identification, Isolation, and Characterization of Human LGR5-positive Colon Adenoma Cells

doi: 10.1101/118034

Figure Lengend Snippet: Relative expression of genes involved in the KEGG pathway “Glycolysis and Gluconeogenesis” between LGR5(+) and LGR5(−) human adenoma cells ( P -value=6.57E-09).

Article Snippet: Flow cytometry spike-in experiments ( ) were performed with 1881 LGR5(+) and 1881 LGR5(−) by mixing at varying proportions and analyzing on a LSRII cytometer (BD Biosciences) before and after magnetic separation with rat monoclonal antibody anti-human LGR5 clone 22H2.8 magnetic bead-conjugate (Miltenyi Biotec), with the allophycocyanin (APC) anti-bead check reagent to recognize the bound magnetic beads (Miltenyi Biotec).

Techniques: Expressing

Journal: BMC Cancer

Article Title: Inverse correlation between PDGFC expression and lymphocyte infiltration in human papillary thyroid carcinomas

doi: 10.1186/1471-2407-9-425

Figure Lengend Snippet: Primers and probes for TaqMan qRT-PCR

Article Snippet: Applied Biosystems Assay On Demand, Hs00234042_m1 , 80 , NM_033016.1.

Techniques: Sequencing, Amplification

Journal: BMC Cancer

Article Title: Inverse correlation between PDGFC expression and lymphocyte infiltration in human papillary thyroid carcinomas

doi: 10.1186/1471-2407-9-425

Figure Lengend Snippet: mRNA expression of PDGF ligands and receptors . mRNA expression of PDGF ligands and receptors calculated relative to mRNA expression levels of the endogenous control ( ACTB ) in A) non-euplastic thyroid tissue (NT, green) and a collection of classical (PTC(diff), blue) and clinically aggressive PTCs (PTC(agg), red). All values were adjusted so that the median value of the NT group was equal to one. Bars indicate median value of each individual group. P-values (below) are from Mann-Whitney t-test between groups, as indicated. B) Relative mRNA expression of all PDGF ligands and receptors in each individual biopsy. PDGFA (red triangle), PDGFB (green triangle), PDGFC (yellow square), PDGFD (brown circle), PDGFRA (magenta diamond) and PDGFRB (blue triangle). All values were adjusted so that the median value of the NT group was equal to one.

Article Snippet: Applied Biosystems Assay On Demand, Hs00234042_m1 , 80 , NM_033016.1.

Techniques: Expressing, Control, MANN-WHITNEY

Journal: BMC Cancer

Article Title: Inverse correlation between PDGFC expression and lymphocyte infiltration in human papillary thyroid carcinomas

doi: 10.1186/1471-2407-9-425

Figure Lengend Snippet: Quantification of PDGF expression level by Taman qRT-PCR and microarray hybridization . Expression profiles of A) PDGFA , B) PDGFB and C) PDGFRA accessed by two different techniques, Taman qRT-PCR (blue square) and cDNA microarray hybridization (magenta square) along the specimens investigated. Line is drawn between points for illustration purposes only. The three different groups of thyroid biopsy specimens are listed below the graph; Non-euplastic thyroid specimen (NT), differentiated PTC specimen (PTC (diff)) and poorly differentiated PTC (PTC (agg)).

Article Snippet: Applied Biosystems Assay On Demand, Hs00234042_m1 , 80 , NM_033016.1.

Techniques: Expressing, Quantitative RT-PCR, Microarray, Hybridization

Journal: BMC cancer

Article Title: Establishment and characterization of a new human pancreatic adenocarcinoma cell line with high metastatic potential to the lung.

doi: 10.1186/1471-2407-10-295

Figure Lengend Snippet: Figure 4 Whole-genome DNA profile of PaCa 5061. The DNA profile performed by Affymetrix GeneChip hybridization shows a considerable num- ber of large chromosomal alterations. Detailed candidate regions of gene amplification and affected genes are listed in Table 1.

Article Snippet: In the present study, we report biomolecular characteristics of a new human pancreatic adenocarcinoma cell line named PaCa 5061, which spontaneously metastasized into the lungs in a mouse xenograft model. We describe the cell line in terms of growth characteristics, phenotype, and genotype for their unique DNA and RNA profile using Affymetrix microarray technology as well as for specific alterations of relevant tumor-associated genes.

Techniques: Hybridization, Amplification

Journal: Frontiers in Veterinary Science

Article Title: Advances in the Diagnosis of Foot-and-Mouth Disease

doi: 10.3389/fvets.2020.00477

Figure Lengend Snippet: Molecular diagnostic assays for detection of FMDV infection.

Article Snippet: , This study compared the performance of two commercially available one step RT-qPCR systems, TaqMan® Fast Virus 1-Step Master Mix (Applied Biosystems®) and Superscript III Platinum® One-Step qRT-PCR Kit (Invitrogen™) in detection of FMDV RNA from milk samples, a non-invasive alternative for detection and typing of FMDV , • The detection limit of Superscript III Platinum® One-Step qRT-PCR Kit and TaqMan® Fast Virus 1-Step Master Mix are 10 −6 and 10 −5 dilutions of FMDV A/KEN/6/2012, respectively , Serum, milk, vesicular epithelium or fluid samples of bovine origin , ( , ) .

Techniques: Diagnostic Assay, Infection, Amplification, Sequencing, Detection Assay, In Vitro, Plasmid Preparation, Multiplex Assay, SYBR Green Assay, In Silico, Produced, Microarray, Hybridization, Cell Culture, RT Lamp Assay

Journal: Nature Communications

Article Title: The oncogene AAMDC links PI3K-AKT-mTOR signaling with metabolic reprograming in estrogen receptor-positive breast cancer

doi: 10.1038/s41467-021-22101-7

Figure Lengend Snippet: a Analysis of somatic alterations of AAMDC using cancer genomic data sets and tools available from cBioPortal (see “Methods”). The frequency of amplification is shown as a percentage and the sample numbers are shown in brackets. METABRIC Molecular Taxonomy of Breast Cancer International Consortium, TCGA The Cancer Genome Atlas, BRCA Breast Cancer, INSERM Institut national de la santé et de la recherche médicale, MBC Metastatic Breast Cancer, NSCLC non-small-cell lung carcinoma, FHCRC Fred Hutchinson Cancer Research Center, NEPC National Environment Protection Council, PanCan Pan-Cancer. b Kaplan–Meier survival plots for patients with tumors expressing high (red) or low (green) levels of AAMDC mRNA. The lower left plots correspond to luminal B tumors treated with tamoxifen (see “Methods”). The p value shown for each plot is determined by the log-rank test. GEO Gene Expression Omnibus, GSE genomic spatial event, NSCLC non-small-cell lung carcinoma. c Localization of the AAMDC protein in tumors from a breast tissue microarray (TMA) assessed by immunohistochemistry (IHC). Representative IHC sections of normal breast tissue, estrogen receptor-negative (ER − ) tumor tissue, ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC) are shown. 0, 1+, 2+, 3+ indicate the staining intensity score. d Associations between AAMDC expression (IHC) and lymph node metastasis (LN + ) as well as tumor grade, tumor size (T3-4), and ER positivity (ER + ) by AAMDC localization from the same TMA. Statistical significance is indicated by Chi-square analysis with a one-tailed p -value relative to ER − tissue. For T3-4: * p = 0.03; for LN + : * p = 0.03; for ER + , from left to right: * p = 0.003, * p = 0.005, * p = 0.005. n = 60 biologically independent samples. Full details of the TMA are provided in Supplementary Table . e Frequency of AAMDC amplification/polysomy in a cohort of 119 luminal B breast cancer specimens. Representative fluorescence in situ hybridization (FISH) images are indicated, with specific probes for AAMDC (red) and Centromere enumeration 11 probe for chromosome 11 ( C11 , green). The full clinical and pathological features of these tumors are shown in Supplementary Data . f Real-time expression analyses (qRT-PCR) of AAMDC in luminal, non-luminal, and normal-like breast cells. Significance levels are determined relative to MCF-12A by Ordinary one-way ANOVA with Dunnett multiple comparison test. Data are presented as mean values ± SEM (* p = 0.0217, ** p = 0.0018, **** p < 0.0001). n = 3 biologically independent RNA extractions. Representative images of immunocytochemistry (ICC) and FISH of selected luminal cell lines are presented. HuMECs non-transformed human mammary epithelial cells.

Article Snippet: For experiments involving non-genetically manipulated human mammary epithelial cells (HuMECs) (Fig. ), human primary mammary epithelial cells were purchased from ATCC (HMEC, PCS-600-010).

Techniques: Amplification, Expressing, Gene Expression, Microarray, Immunohistochemistry, In Situ, Staining, One-tailed Test, Fluorescence, In Situ Hybridization, Quantitative RT-PCR, Comparison, Immunocytochemistry, Transformation Assay